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Figure 6


Fig. 6. FAP133 associates with other IFT proteins in a large macromolecular complex. (A) Flagella matrix components were fractionated using a Superose-6 gel filtration column. The eluted fractions were separated in two 5-15% polyacrylamide gels and stained with Coomassie Blue (upper panel); similar gels were transferred to nitrocellulose and probed with the indicated antibodies (lower panels). A significant amount of FAP133 was found in fraction 2, co-purifying with DHC1b-D1bLIC as well as clear peaks of LC8, FLA10 and the complex A protein IFT139. This fraction contained particles of >~2 MDa as outer arm dynein components (e.g. LC2) eluted in later fractions. Note that the LC8 peak is more spread out towards later fractions as this protein is also an integral component of the outer dynein arm and of inner arm I1/f. (B) Protein G-agarose beads which were previously treated with antibody against FAP133 ({alpha}-FAP133) or BSA alone (control), were incubated with flagella matrix extracts, and proteins present in the pellet and extract (10% input) were identified by immunoblotting using the indicated antibodies against various IFT proteins. An antibody against IC1 which is an outer-dynein-arm component (see DiBella and King, 2001) was included as a negative control. (C) In a similar experiment, flagellar matrix extracts were incubated with an antibody against IFT139 ({alpha}-IFT139) or an equivalent volume of PBS (control) followed by incubation with protein-G–agarose beads. The immunoprecipitated pellets were analyzed for the presence of IFT proteins by immunoblotting. The antibody against EB1 [a plus-end microtubule binding protein that localizes to the flagellar tip and basal bodies (Pedersen et al., 2003)] was used as a negative control. (D) The complex B protein IFT172 was immunoprecipitated from a flagellar extract using the anti-IFT172 antibody ({alpha}-IFT172); the control sample contained BSA alone. Only another complex B protein (IFT81) and a small amount of IFT139 from complex A were found in the pellet; DHC1b, FAP133, FLA10 and outer arm IC1 were not detected.